The complement system is a complex enzyme cascade made up of a series of serum glycoproteins, that normally exist in inactive, pro-enzyme form. Two main pathways, the classical and the alternative pathway, can activate complement, which merge at the level of C3, where two similar C3 convertases cleave C3 into C3a and C3b.
Macrophages are specialist cells that have developed an innate capacity to recognize subtle differences in the structure of cell-surface expressed identification tags, so called molecular patterns (Taylor, et al., Eur J Immunol 33, 2090-2097 (2003); Taylor, et al. Annu Rev Immunol 23, 901-944 (2005)). While the direct recognition of these surface structures is a fundamental aspect of innate immunity, opsonization allows generic macrophage receptors to mediate engulfment, increasing the efficiency and diversifying recognition repertoire of the phagocyte (Stuart and Ezekowitz, Immunity 22, 539-550 (2005)). The process of phagocytosis involves multiple ligand-receptor interactions, and it is now clear that various opsonins, including immunoglobulins, collectins, and complement components, guide the cellular activities required for pathogen internalization through interaction with macrophage cell surface receptors (reviewed by Aderem and Underhill, Annu Rev Immunol 17, 593-623 (1999); Underhill and Ozinsky, Annu Rev Immunol 20, 825-852 (2002)). While natural immunoglobulins encoded by germline genes can recognize a wide variety of pathogens, the majority of opsonizing IgG is generated through adaptive immunity, and therefore efficient clearance through Fc receptors is not immediate (Carroll, Nat Immunol 5, 981-986 (2004)). Complement, on the other hand, rapidly recognizes pathogen surface molecules and primes the particle for uptake by complement receptors (Brown, Infect Agents Dis 1, 63-70 (1991)).
Complement consists of over 30 serum proteins that opsonize a wide variety of pathogens for recognition by complement receptors. Depending on the initial trigger of the cascade, three pathways can be distinguished (reviewed by (Walport, N Engl J Med 344, 1058-1066 (2001)). All three share the common step of activating the central component C3, but they differ according to the nature of recognition and the initial biochemical steps leading to C3 activation. The classical pathway is activated by antibodies bound to the pathogen surface, which in turn bind the C1q complement component, setting off a serine protease cascade that ultimately cleaves C3 to its active form, C3b. The lectin pathway is activated after recognition of carbohydrate motifs by lectin proteins. To date, three members of this pathway have been identified: the mannose-binding lectins (MBL), the SIGN-R1 family of lectins and the ficolins (Pyz et al., Ann Med 38, 242-251 (2006)) Both MBL and ficolins are associated with serine proteases, which act like C1 in the classical pathway, activating components C2 and C4 leading to the central C3 step. The alternative pathway contrasts with both the classical and lectin pathways in that it is activated due to direct reaction of the internal C3 ester with recognition motifs on the pathogen surface. Initial C3 binding to an activating surface leads to rapid amplification of C3b deposition through the action of the alternative pathway proteases Factor B and Factor D. Importantly, C3b deposited by either the classical or the lectin pathway also can lead to amplification of C3b deposition through the actions of Factors B and D. In all three pathways of complement activation, the pivotal step in opsonization is conversion of the component C3 to C3b. Cleavage of C3 by enzymes of the complement cascades exposes the thioester to nucleophilic attack, allowing covalent attachment of C3b onto antigen surfaces via the thioester domain. This is the initial step in complement opsonization. Subsequent proteolysis of the bound C3b produces iC3b, C3c and C3dg, fragments that are recognized by different receptors (Ross and Medof, Adv Immunol 37, 217-267 (1985)). This cleavage abolishes the ability of C3b to further amplify C3b deposition and activate the late components of the complement cascade, including the membrane attack complex, capable of direct membrane damage. However, macrophage phagocytic receptors recognize C3b and its fragments preferentially; due to the versatility of the ester-bond formation, C3-mediated opsonization is central to pathogen recognition (Holers et al., Immunol Today 13, 231-236 (1992)), and receptors for the various C3 degradation products therefore play an important role in the host immune response.
C3 itself is a complex and flexible protein consisting of 13 distinct domains. The core of the molecule is made up of 8 so-called macroglobulin (MG) domains, which constitute the tightly packed α and β chains of C3. Inserted into this structure are CUB (Clr/Cls, Uegf and Bone mophogenetic protein-1) and TED domains, the latter containing the thioester bond that allows covalent association of C3b with pathogen surfaces. The remaining domains contain C3a or act as linkers and spacers of the core domains. Comparison of C3b and C3c structures to C3 demonstrate that the molecule undergoes major conformational rearrangements with each proteolysis, which exposes not only the TED, but additional new surfaces of the molecule that can interact with cellular receptors (Janssen and Gros, Mol Immunol 44, 3-10 (2007)).
In order to prevent unwanted complement activation, most mammalian cells are equipped with regulators that block complement amplification on host self cells (Hourcade et al. Adv Immunol 45:381 (1989)). In the absence of these intrinsic regulators, serum exposure results in the generation of complement split product that in turn facilitate inflammation and tissue damage (Oglesby et al. J Exp Med 175:1547 (1992) and Oglesby et al., Trans Assoc. Am. Physicians 104:164 (1991)). Non-cellular surfaces that lack intrinsic complement regulators are therefore especially prone to complement attack and are fully dependent on protection by soluble complement regulators in serum. Uncontrolled complement activation due to the lack of appropriate complement regulation has been associated with various chronic inflammatory diseases and degenerative diseases. Dominant in this inflammatory cascade are the complement split products C3a and C5a that function as chemo-attractant and activators of neutrophils and inflammatory macrophages via the C3a and C5a receptors (Mollnes et al., Trends Immunol 23:61 (2002)). Properdin, released from neutrophils, further amplifies the inflammatory cascade through stabilization of the AP convertase (Lutz and Jelezarova, Mol. Immunol. 43:2 (2006)). Complement activation has been shown to be an important component driving inflammation in immune-complex mediated diseases such as membranoproliferative glomerulonephritis, nephrotoxic nephritis and arthritis (Walport, N. Engl. J. Med. 344:1058 (2001); Thurman and Holers, J. Immunol. 176:1305 (2006); Banda et al., J. Immunol. 171:2109 (2003); Weisman et al., Science 249:146 (1990); Morgan and Harris, Mol. Immunol. 40:159 (2003)), as well as age-related macular degeneration (Anderson et al., Am. J. Ophthalmol. 134:411 (2002); Donoso et al., Surv. Ophthalmol. 51:137 (2006); Gold et al., Natl. Genet. 38:458 (2006); Hageman et al., Proc. Natl. Acad. Sci. USA 102:7227 (2005); Hageman et al., Ann. Med. 38:592 (2006); Hageman et al., Prog. Retin. Eye Res. 20:705 (2001)).
Most regulators of complement activation act at the level of C3b, the central component of the complement convertases. These natural regulators of complement activation are typically large in size (>100 kDa) and difficult to develop as a therapeutic reagent. Accordingly, there is a need for therapeutic agents to prevent and treat complement-associated disorders by blocking C3b.